Metabolic and transcriptomic characterization of summer and winter dormancy in the solitary bee, Osmia lignaria.

The solitary bee Osmia lignaria is a native pollinator in North America with growing economic importance. The life cycle of O. lignaria provides a unique opportunity to compare the physiological and molecular mechanisms underlying two ecologically contrasting dormancies within the same species. O. lignaria prepupae become dormant during the summer to avoid high temperatures. Shortly after adult eclosion, they enter a second dormancy and overwinter as diapausing adults. To compare these two dormancies, we measured metabolic rates and gene expression across development as bees initiate, maintain, and terminate both prepupal (summer) and adult (overwintering) dormancies. We observed a moderate temperature-independent decrease in gas exchange during both the prepupal dormancy after cocoon spinning (45 %) and during adult diapause after eclosion (60 %). We sequenced and assembled a high-quality reference genome from a single haploid male bee with a contiguous n50 of 5.5 Mbp to facilitate our transcriptomic analysis. The transcriptomes of dormant prepupae and diapausing adults clustered into distinct groups more closely associated with life stage than dormancy status. Membrane transport, membrane-bound cellular components, oxidoreductase activity, glutathione metabolism, and transcription factor activity increased during adult diapause, relative to prepupal dormancy. Further, the transcriptomes of adults in diapause clustered into two groups, supporting multiple phases of diapause during winter. Late adult diapause was associated with gene expression profiles supporting increased insulin/IGF, juvenile hormone, and ecdysone signaling.

Molecular mechanisms and trade-offs underlying fluctuating thermal regimes during low-temperature storage.

Insects exposed to constant low temperatures (CLT) exhibit high rates of mortality as well as a variety of sublethal effects. In many species, interruptions of CLT with brief pulses of warm temperatures (fluctuating thermal regimes, FTR) lead to increases in survival and fewer sublethal effects. However, we still lack a complete understanding of the physiological mechanisms activated during FTR. In this review, we discuss recent advances in understanding FTR's underlying molecular mechanisms. We discuss knowledge gaps related to potential trade-offs between FTR's beneficial effects and the costs of these repairs to overwintering reserves and reproduction. We present the hypothesis that the warm pulse of FTR helps to maintain daily rhythmicity.

Nesting cavity diameter has implications for management of the alfalfa leafcutting bee (Hymenoptera: Megachilidae).

Body size influences performance in many bee species and may be influenced by nesting cavity diameter in cavity-nesting bees. Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae) is a commercially-managed, solitary cavity-nesting bee. In M. rotundata body size has low heritability and is strongly influenced by the size of the larval provision and the diameter of the nesting cavity. Commercial nesting boxes have cavities that are 7 mm in diameter. Our goal was to examine the effects that nesting cavity diameter has on M. rotundata body size and performance by manipulating the size of cavities that are available for nesting. We provided bees with nesting cavities that ranged in size from 4 to 9 millimeters in 1 mm increments. To assess body size we measured mass and intertegular span. To assess performance we measured wing area, wing loading, sex, overwintering survival, pollen ball occurrence, and diapause status in the offspring. We also examined the reproductive output from the different nest cavity diameters. We found that the 8 mm cavities reared bees with the largest mass, and 4 mm cavities reared bees with the smallest mass. We determined that the 7 mm nesting cavity is optimal for offspring yield, the 8 mm nesting cavity is optimal for performance, and the 5 mm nesting cavity may be optimal for conservation efforts of other cavity-nesting bees. Based on the desired outcome of the bee managers, nest sizes differing from the standard may provide an advantage.

Effects of Temperature and Wildflower Strips on Survival and Macronutrient Stores of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae) Under Extended Cold Storage.

Megachile rotundata (F.) is an important pollinator of alfalfa in the United States. Enhancing landscapes with wildflowers is a primary strategy for conserving pollinators and may improve the sustainability of M. rotundata. Changing cold storage temperatures from a traditionally static thermal regime (STR) to a fluctuating thermal regime (FTR) improves overwintering success and extends M. rotundata's shelf life and pollination window. Whether floral resources enhance overwintering survival and/or interact with a thermal regime are unknown. We tested the combined effects of enhancing alfalfa fields with wildflowers and thermal regime on survival and macronutrient stores under extended cold storage (i.e., beyond one season). Megachile rotundata adults were released in alfalfa plots with and without wildflower strips. Completed nests were harvested in September and stored in STR. After a year, cells were randomly assigned to remain in STR for 6 months or in FTR for a year of extended cold storage; emergence rates were observed monthly. Macronutrient levels of emerged females were assessed. FTR improved M. rotundata survival but there was no measurable effect of wildflower strips on overwintering success or nutrient stores. Timing of nest establishment emerged as a key factor: offspring produced late in the season had lower winter survival and dry body mass. Sugars and glycogen stores increased under FTR but not STR. Trehalose levels were similar across treatments. Total lipid stores depleted faster under FTR. While wildflowers did not improve M. rotundata survival, our findings provide mechanistic insight into benefits and potential costs of FTR for this important pollinator.

Environmental impacts on diapause and survival of the alfalfa leafcutting bee, Megachile rotundata.

Megachile rotundata exhibits a facultative prepupal diapause but the cues regulating diapause initiation are not well understood. Possible cues include daylength and temperature. Megachile rotundata females experience changing daylengths over the nesting season that may influence diapause incidence in their offspring through a maternal effect. Juvenile M. rotundata spend their developmental period confined in a nesting cavity, potentially subjected to stressful temperatures that may affect diapause incidence and survival. To estimate the impact of daylength and nest cavity temperature on offspring diapause, we designed a 3D printed box with iButtons that measured nest cavity temperature. We observed nest building throughout the season, monitored nest cavity temperature, and followed offspring through development to measure diapause incidence and mortality. We found that daylength was a cue for diapause, and nest cavity temperature did not influence diapause incidence. Eggs laid during long days had a lower probability of diapause. Siblings tended to have the same diapause status, explaining a lot of the remaining variance in diapause incidence. Some females established nests that contained both diapausing and nondiapausing individuals, which were distributed throughout the nest. Nest cavities reached stressful temperatures, which decreased survival. Mortality was significantly higher in nondiapausing bees and the individuals that were laid first in the nest. In conclusion, we demonstrate a maternal effect for diapause that is mediated by daylength and is independent of nest box temperature.

Fluctuating temperatures extend longevity in pupae and adult stages of the sepsid Themira biloba.

Fluctuating Thermal Regimes (FTR), where organisms are held at low temperatures with a brief, daily warm pulse, have been shown to increase longevity in adult insects and improve pupa survival while reducing sublethal effects. We used FTR to extend the longevity and thus generation time of the fly species Themira biloba (Diptera: Sepsidae). T. biloba can be maintained in continuous culture and requires an insecticide-free dung substrate for larval growth and development. Our objective was to decrease labor and consumable materials required to maintain insect species in critical scientific collections using FTR. We extended pupation time from 4 days up to 8 weeks with no increase in mortality, and mean adult longevity was increased from 12 days to 50 days. FTR is a valuable tool for reducing the investment required to maintain rare and exotic insects.

Body size allometry impacts flight-related morphology and metabolic rates in the solitary bee Megachile rotundata.

Body size is related to many aspects of life history, including foraging distance and pollination efficiency. In solitary bees, manipulating the amount of larval diet produces intraspecific differences in adult body size. The goal of this study was to determine how body size impacts metabolic rates, allometry, and flight-related morphometrics in the alfalfa leafcutting bee, Megachile rotundata. By restricting or providing excess food, we produced a range of body sizes, which allowed us to test the effect of body size on allometry, the power required for flight, and amount of energy produced, as measured indirectly through CO2 emission. The power required during flight was predicted using the flight biomechanical formulas for wing loading and excess power index. We found larger bees had higher absolute metabolic rates at rest and during flight, but smaller bees had higher mass-specific metabolic rates at rest. During flight, bees did not have size-related differences in mass-specific metabolic rate. As bees increase in size, their thorax and abdomens become disproportionately larger, while their wings (area, and length) become disproportionately smaller. Smaller bees had more power available during flight as demonstrated by flight biomechanical formulas. Smaller body size was advantageous because of a reduced power requirement for flight with no metabolic cost.

Body and Wing Allometries Reveal Flight-Fecundity Tradeoff in Response to Larval Provisioning in Osmia lignaria (Hymenoptera: Megachilidae).

Variation in body size has important implications for physical performance and fitness. For insects, adult size and morphology are determined by larval growth and metamorphosis. Female blue orchard bees, Osmia lignaria, (Say) provision a finite quantity of food to their offspring. In this study, we asked how provision-dependent variation in size changes adult morphology. We performed a diet manipulation in which some larvae were starved in the final instar and some were given unlimited food. We examined the consequences on adult morphology in two ways. First, allometric relationships between major body regions (head, thorax, abdomen) and total body mass were measured to determine relative growth of these structures. Second, morphometrics that are critical for flight (wing area, wing loading, and extra flight power index) were quantified. Head and thorax mass had hyperallometric relationships with body size, indicating these parts become disproportionately large in adults when larvae are given copious provisions. However, abdominal mass and wing area increased hypoallometrically with body size. Thus, large adults had disproportionately lighter abdomens and smaller wing areas than smaller adults. Though both males and females followed these general patterns, allometric patterns were affected by sex. For flight metrics, small adults had reduced wing loading and an increased extra flight power index. These results suggest that diet quantity alters development in ways that affect the morphometric trait relationships in adult O. lignaria and may lead to functional differences in performance.

Diet quantity influences caste determination in honeybees (Apis mellifera).

In species that care for their young, provisioning has profound effects on offspring fitness. Provisioning is important in honeybees because nutritional cues determine whether a female becomes a reproductive queen or sterile worker. A qualitative difference between the larval diets of queens and workers is thought to drive this divergence; however, no single compound seems to be responsible. Diet quantity may have a role during honeybee caste determination yet has never been formally studied. Our goal was to determine the relative contributions of diet quantity and quality to queen development. Larvae were reared in vitro on nine diets varying in the amount of royal jelly and sugars, which were fed to larvae in eight different quantities. For the middle diet, an ad libitum quantity treatment was included. Once adults eclosed, the queenliness was determined using principal component analysis on seven morphological measurements. We found that larvae fed an ad libitum quantity of diet were indistinguishable from commercially reared queens, and that queenliness was independent of the proportion of protein and carbohydrate in the diet. Neither protein nor carbohydrate content had a significant influence on the first principle component 1 (PC1), which explained 64.4% of the difference between queens and workers. Instead, the total quantity of diet explained a significant amount of the variation in PC1. Large amounts of diet in the final instar were capable of inducing queen traits, contrary to the received wisdom that queen determination can only occur in the third instar. These results indicate that total diet quantity fed to larvae may regulate the difference between queen and worker castes in honeybees.

Microclimate Temperatures Impact Nesting Preference in Megachile rotundata (Hymenoptera: Megachilidae).

The temperature of the nest influences fitness in cavity-nesting bees. Females may choose nest cavities that mitigate their offspring's exposure to stressful temperatures. This study aims to understand how cavity temperature impacts the nesting preference of the solitary bee Megachile rotundata (Fabricius) under field conditions. We designed and 3D printed nest boxes that measured the temperatures of 432 cavities. Nest boxes were four-sided with cavity entrances facing northeast, northwest, southeast, and southwest. Nest boxes were placed along an alfalfa field in Fargo, ND and were observed daily for completed nests. Our study found that cavity temperature varied by direction the cavity faced and by the position of the cavity within the nest box. The southwest sides recorded the highest maximum temperatures while the northeast sides recorded the lowest maximum temperatures. Nesting females filled cavities on the north-facing sides faster than cavities on the south-facing sides. The bees preferred to nest in cavities with lower average temperatures during foraging hours, and cavities that faced to the north. The direction the cavity faced was associated with the number of offspring per nest. The southwest-facing cavities had fewer offspring than nests on the northeast side. Our study indicates that the nesting box acts as a microclimate, with temperature varying by position and direction of the cavity. Variation in cavity temperature affected where females chose to nest, but not their reproductive investment.

Fluctuating thermal regimes prevent chill injury but do not change patterns of oxidative stress in the alfalfa leafcutting bee, Megachile rotundata.

In insects, prolonged exposure to unseasonably low temperatures can lead to detrimental physiological effects known as chill injury. Changes to active and passive transport across epithelia during chilling likely drive the collapse of ion gradients, metabolic imbalance and potentially oxidative stress. In the alfalfa leafcutting bee, Megachile rotundata transcriptomic evidence provides support for these responses at the level of gene expression, but variable expression profiles between life stages in M. rotundata indicate that different mechanisms could be responsible for repairing and protecting against chill injuries across development. Herein, we test the hypotheses that 1) chill injury leads to oxidative stress and damage in insects and 2) exposure to a fluctuating thermal regime (FTR) promotes an increased oxidative stress response leading to a decrease in damage by reactive oxygen species. We measured the expression of transcripts with products known to have antioxidant properties in overwintering prepupae as well as total antioxidant capacity and lipid peroxidation during both extended overwintering in prepupae and low temperature stress during pupal development. We observed differential gene expression for the antioxidant glutathione peroxidase and several transcripts with putative antioxidant properties including vitellogenin, apolipoprotein D, glutathione S-transferase, and nuclear protein 1. However, the expression of transcripts coding for other enzymatic antioxidants did not change between treatments. Neither life stage varied in their capacity to cope with an induced oxidative stress after FTR exposure and we did not observe evidence of lipid peroxidation in chill injured (STR) prepupae. These results did not support our initial hypotheses and indicate that oxidative-stress-induced damage is neither a causal factor or symptom of chill injury.

Thermoprofile Parameters Affect Survival of Megachile rotundata During Exposure to Low-Temperatures.

Insects exposed to low temperature stress can experience chill injury, but incorporating fluctuating thermoprofiles increases survival and blocks the development of sub-lethal effects. The specific parameters required for a protective thermoprofile are poorly understood, because most studies test a limited range of thermoprofiles. For example, thermoprofiles with a wave profile may perform better than a square profile, but these two profiles are rarely compared. In this study, two developmental stages of the alfalfa leafcutting bee, Megachile rotundata, eye-pigmented pupae, and emergence-ready adults, were exposed to one of eight thermoprofiles for up to 8 weeks. All the thermoprofiles had a base of 6°C and a peak temperature of either 12°C or 18°C. The duration at peak temperature varied depending on the shape of the thermoprofile, either square or wave form. Two other treatments acted as controls, a constant 6°C and a fluctuating thermal regime (FTR) with a base temperature of 6°C that was interrupted daily by a single, 1-h pulse at 20°C. Compared with constant 6°C, all the test thermoprofiles significantly improved survival. Compared with the FTR control, the thermoprofiles with a peak temperature of 18°C outperformed the 12°C profiles. Bees in the eye-pigmented stage exposed to the 18°C profiles separated into two groups based on the shape of the profile, with higher survival in the square profiles compared with the wave profiles. Bees in the emergence-ready stage exposed to 18°C profiles all had significantly higher survival than bees in the FTR controls. Counter to expectations, the least ecologically relevant thermoprofiles (square) had the highest survival rates and blocked the development of sub-lethal effects (delayed emergence).

Immediate Transcriptional Response to a Temperature Pulse under a Fluctuating Thermal Regime.

The response of ectotherms to temperature stress is complex, non-linear, and is influenced by life stage and previous thermal exposure. Mortality is higher under constant low temperatures than under a fluctuating thermal regime (FTR) that maintains the same low temperature but adds a brief, daily pulse of increased temperature. Long term exposure to FTR has been shown to increase transcription of genes involved in oxidative stress, immune function, and metabolic pathways, which may aid in recovery from chill injury and oxidative damage. Previous research suggests the transcriptional response that protects against sub-lethal damage occurs rapidly under exposure to fluctuating temperatures. However, existing studies have only examined gene expression after a week or over many months. Here we characterize gene expression during a single temperature cycle under FTR. Development of pupating alfalfa leafcutting bees (Megachile rotundata) was interrupted at the red-eye stage and were transferred to 6°C with a 1-h pulse to 20°C and returned to 6°C. RNA was collected before, during, and after the temperature pulse and compared to pupae maintained at a static 6°C. The warm pulse is sufficient to cause expression of transcripts that repair cell membrane damage, modify membrane composition, produce antifreeze proteins, restore ion homeostasis, and respond to oxidative stress. This pattern of expression indicates that even brief exposure to warm temperatures has significant protective effects on insects exposed to stressful cold temperatures that persist beyond the warm pulse. Megachile rotundata's sensitivity to temperature fluctuations indicates that short exposures to temperature changes affect development and physiology. Genes associated with developmental patterning are expressed after the warm pulse, suggesting that 1 h at 20°C was enough to resume development in the pupae. The greatest difference in gene expression occurred between pupae collected after the warm pulse and at constant low temperatures. Although both were collected at the same time and temperature, the transcriptional response to one FTR cycle included multiple transcripts previously identified under long-term FTR exposure associated with recovery from chill injury, indicating that the effects of FTR occur rapidly and are persistent.

Long-Distance Transportation Causes Temperature Stress in the Honey Bee, Apis mellifera (Hymenoptera: Apidae).

Pollination services provided by the honey bee, Apis mellifera (Hymenoptera: Apidae, Linnaeus, 1758) have broad economic impacts and are necessary for production of a diversity of important crops. Hives may be transported multiple times per year to provide pollination. To test how temperature may contribute to transportation stress, temperature sensors were placed in hives in different locations and orientations on the trailer during shipping. Colony size prior to shipping significantly contributed to loss of population immediately after shipping which contributed to colony failure with smaller colonies more likely to fail and fail faster. Colony size also affects thermoregulation and temperature stress. Internal hive temperature varies significantly based on location and orientation. While colonies near the front and rear of the trailer and those oriented toward the center aisle had significantly different average internal temperatures, colony size best predicts loss of thermoregulation. Additionally, we profiled gene expression at departure, on arrival, and after a recovery period to identify transcriptional responses to transportation. Functional and enrichment analysis identified increased methylation and decreased ribosomal and protein-folding activity. Pheromone and odorant-binding transcripts were up-regulated after transportation. After recovery, transcripts associated with defense response, immune activity, and heat shock decreased, while production of antibiotic peptides increased. We conclude that hives experience considerable temperature stress possibly caused by turbulent airflow in exposed locations. Transportation stress should be considered an important component of annual colony losses which can be mitigated with improved management strategies.

Comparative analysis reveals the complex role of histoblast nest size in the evolution of novel insect abdominal appendages in Sepsidae (Diptera).

BACKGROUND: The males of some sepsid species (Sepsidae: Diptera) have abdominal appendages that are remarkable in several ways. They are sexually dimorphic, have a complex evolutionary history of gain and loss, and can be jointed and thus highly mobile. The sternite brushes are used extensively in complex courtship behaviors that differ considerably between species and during mating. The abdominal appendages have a novel developmental pathway developing from histoblast nests rather than imaginal discs. RESULTS: We focus on the evolution of cell number, nest area, and segment length in both sexes to understand how this tissue relates to the formation of novel abdominal appendages. We map histoblast nest size of wandering-phase larvae of 17 species across 10 genera to a phylogenetic tree of Sepsidae and demonstrate that abdominal appendages require significant increases of histoblast nest size and cell number in most species while one species produces small appendages even without such modifications. In species with particularly large appendages, not only the nests on the fourth, but nests in neighboring segments are enlarged (Themira biloba, Themira putris). The loss of abdominal appendages corresponds to the loss of an enlarged fourth histoblast nest, although one species showed an exception to this pattern. One species that constitutes an independent origin of abdominal appendages (Perochaeta dikowi) uses an unusual developmental mechanism in that the histoblast nest sizes are not sexually dimorphic. CONCLUSIONS: The surprisingly high diversity in histoblast size and degree of sexual dimorphism suggests that the developmental mechanism used for abdominal appendage formation in sepsids is highly adaptable. The presence of appendages usually correlate with increased histoblast cell number and in most cases appendage loss results in a return to ancestral histoblast morphology. However, we also identify several exceptions that indicate the abdominal appendages have a malleable developmental origin that is responsive to selection.

Micro-computed tomography of pupal metamorphosis in the solitary bee Megachile rotundata.

Insect metamorphosis involves a complex change in form and function. In this study, we examined the development of the solitary bee, Megachile rotundata, using micro-computed tomography (µCT) and volume analysis. We describe volumetric changes of brain, tracheae, flight muscles, gut, and fat bodies in prepupal, pupal, and adult M. rotundata. We observed that individual organ systems have distinct patterns of developmental progression, which vary in their timing and duration. This has important implications for commercial management of this agriculturally relevant pollinator.

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata.

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA sequencing (RNA-seq). However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory-reared and -maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata, entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory- and field-maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect.

Phenotypic integration in an extended phenotype: among-individual variation in nest-building traits of the alfalfa leafcutting bee (Megachile rotundata).

Structures such as nests and burrows are an essential component of many organisms' life-cycle and require a complex sequence of behaviours. Because behaviours can vary consistently among individuals and be correlated with one another, we hypothesized that these structures would (1) show evidence of among-individual variation, (2) be organized into distinct functional modules and (3) show evidence of trade-offs among functional modules due to limits on energy budgets. We tested these hypotheses using the alfalfa leafcutting bee, Megachile rotundata, a solitary bee and important crop pollinator. Megachile rotundata constructs complex nests by gathering leaf materials to form a linear series of cells in pre-existing cavities. In this study, we examined variation in the following nest construction traits: reproduction (number of cells per nest and nest length), nest protection (cap length and number of leaves per cap), cell construction (cell size and number of leaves per cell) and cell provisioning (cell mass) from 60 nests. We found a general decline in investment in cell construction and provisioning with each new cell built. In addition, we found evidence for both repeatability and plasticity in cell provisioning with little evidence for trade-offs among traits. Instead, most traits were positively, albeit weakly, correlated (r ~ 0.15), and traits were loosely organized into covarying modules. Our results show that individual differences in nest construction are detectable at a level similar to that of other behavioural traits and that these traits are only weakly integrated. This suggests that nest components are capable of independent evolutionary trajectories.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Metamorphosis is induced by food absence rather than a critical weight in the solitary bee, Osmia lignaria.

Body size is an important phenotypic trait that correlates with performance and fitness. For determinate growing insects, body size variation is determined by growth rate and the mechanisms that stop growth at the end of juvenile growth. Endocrine mechanisms regulate growth cessation, and their relative timing along development shapes phenotypic variation in body size and development time. Larval insects are generally hypothesized to initiate metamorphosis once they attain a critical weight. However, the mechanisms underlying the critical weight have not been resolved even for well-studied insect species. More importantly, critical weights may or may not be generalizable across species. In this study, we characterized the developmental aspects of size regulation in the solitary bee, Osmia lignaria We demonstrate that starvation cues metamorphosis in O. lignaria and that a critical weight does not exist in this species. Larvae initiated pupation <24 h after food was absent. However, even larvae fed ad libitum eventually underwent metamorphosis, suggesting that some secondary mechanism regulates metamorphosis when provisions are not completely consumed. We show that metamorphosis could be induced by precocene treatment in the presence of food, which suggests that this decision is regulated through juvenile hormone signaling. Removing food at different larval masses produced a 10-fold difference in mass between smallest and largest adults. We discuss the implications of body size variation for insect species that are provided with a fixed quantity of provisions, including many bees which have economic value as pollinators.